J . Med. Chem. 1992,35,1273-1279
1273
Synthesis and Substance P Antagonist Activity of Naphthimidazolium Derivatives Kristine B. Lawrence, Bhaskar R. Venepalli,* Kenneth C. Appell, Ramanuj Goswami, Margaret E. Logan, Bruce E. Tomczuk, and John M. Yanni Life Sciences Research Laboratories, Eastman Kodak Company, Rochester, New York 14650-2158. Received May 17, 1991 The synthesis of unsymmetrical naphth[2,3-d]imidazoliumand bridged naphth[2,3-d]imidazoliumderivatives and their substance P (SP)antagonist activity are described. All compounds were evaluated for their ability to displace SP from neurokinin-1 (NK-1) receptor sites using standard receptor binding methodology (rat forebrain membrane). 2,3-d]imidazolium chloride 1,3-Diethyl-2-[3-(l,3-dihydro-1,3,3-trimethyl-W-indol-2-ylidene)-l-propenyl]-~--naphth[ (?a), a representative compound in this series, waa further evaluated for SP antagonist activity in a guinea pig ileum contractility assay. In vivo SP antagonist activity of 7a was demonstrated using SP-induced salivation and paw edema models performed in rata.
Introduction Substance P (SP)is an undecapeptide neurotransmitter and is implicated in several important disease states, particularly in pain and inflammation.’P2 However, the therapeutic significance of a SP antagonist has not been fuuy explored, primarily due to the lack of orally available, nonpeptide SP antagonists except for the recently disclosed nonpeptidic quinuclidines.3 During ongoing efforts to discover novel, nonpeptide SP antagonists, we have prepared several imidazo[4,5-b]quinoxalinederivatives: represented by general structure I (Figure 1). With a goal to prepare more potent SP antagonists and to understand the structureactivity relationship of these compounds, we were interested in the synthesis of the more electron donating naphth[2,3-d]imidazolium ring system represented by general structure I1 (Figure 1).The target compounds I1 also serve as carbon isosteres of I. The preparation of these compounds and their biological activities as SP antagonists are described here. Chemistry. A series of unsymmetrical compounds containing the unbridged, naphth[2,3-d]imidazole nucleus were prepared from l-ethyl-2-methyl-lH-naphth[2,3-d]imidazole (3, Scheme I, Table I). This heterocycle (3) was obtained by the condensation of 2,3-diaminonaphthalene (1)with triethyl orthoacetate in NJV-dimethylformamide to obtain 2 followed by alkylation with ethyl iodide. Quaternization was accomplished by heating the monosubstituted l€€-naphth[2,&d]imidazole 3 with the desired alkyl halide, yielding either the 1,3-diethyl4aor the 1-ethyl 3-hydroxyethyl4b quaternary salt. The condensation of these quaternary salts with an acetanilidovinylintermediate6i7(5) was achieved either with dry pyridineltriethylamine*or acetonitrile/DBU to give quaternary iodide salts 6a-a (Scheme I, Table I). In an effort to improve solubility, several of these compounds were converted to the corresponding chloride salts (7a-d, Scheme I, Table I) using an ion-exchange resin. Attempts to condense l-ethyl-3-(2-hydroxyethyl)-2methyl-lH-naphth[2,3-d]imidazoliumiodide (4b) with acetanilidovinyl intermediate 5 using the conditions outlined in Scheme I were unsuccessful. Condensation occurred only after 4b was acylated in situ using acetic anhydride, yielding the acylated quaternary salt 6c (Table I). Partial hydrolysis occurred during ion exchange, yielding a 2:l mixture of acylated and nonacylated quaternary chloride salts (7c and 7d, respectively, Table I) which were separated by fractional crystallization. Preparation of 1,3-diethyl-2-[34-methylbenz [cd]indol-2(1H)-ylidene)-l-propenyl]-1H-naphth[2,3-d]imidazolium chloride (9) involved condensation of 4a with
* To whom correspondence should be addressed at Eastman Fine Chemicals, Eastman Kodak Company, 1001 Lee Road, Rochester, NY 14652-3512. 0022-262319211835-1273$03.00/0
[1-methylbenz[cd]indol-2(Vi)-ylidene]acetaldehyde”” (8) in refluxing acetic anhydride followed by ion exchange (Scheme I, Table I). Quaternary derivatives containing the alkyl-bridged naphth[2,3-d]imidazolenucleus (13a-q Scheme 11,Table 11) were prepared by reacting either a 1,2,3,4-tetrahydrolH-naphth[2’,3’:4,5]imidazo[1,2-a]pyridinium or a 2,3dihydro-lH-naphth0[2,3-d]pyrrolo[ 1,2-a]imidazolium quaternary salt (12) with the desired acetanilidovinyl intermediateep75 using triethylamine in dry pyridine or ethanol.I2 The requisite bridged naphth[2,3-d]imidazole quaternary salts 12 were prepared by condensation of 2,3-diaminonaphthalene 1 with the appropriate 6 or ysubstituted lactone1310 followed by quaternization using (1) Regoli, D.; Drapeau, G.; Dion, S.; Couture, R. New Selective Agonists for Neurokinin Receptors: Pharmacological Toole for Receptor Characterization. Trends Pharmacol. Sci. 1988,9, 290-295. (2) otsuko, M.; Yanagisawa, M. Effect of a Tachykinin Antagonist on a Nociceptive Reflex in the Isolated Spinal Cord-Tail Preparation of the New-born Rat. J. Physiol. (London) 1988, 395, 255-270. (3) Snider, R. M.; Constantine, J. W.; Lowe, J. A., III; Longo, K. P.; Lebel, W. S.; Woody, H. A.; Drozda, S. E.; Desai, M. C.; Vinick, F. J.; Spencer, R. W.;H a , H.-J. A Potent Nonpeptidic Antagonist of the Substance P (NK1)Receptor. Science 1991, 435-437. (4) Appell, K. C.; Babb, B. E.; Gcswami, R.; Hall, P. L.; Lawrence, K. B.; Logan, M. E.; Przyklek-Elling, R.; Tomczuk, B. E.; Venepalli, B. R.; Yanni, J. M. Imidazo[4,5-b]quinoxaliieCyanines as Neurokinin Antagonista. J. Med. Chem. 1991,34, 1751-1753. (5) James, T.H.Sensitizing and DesensitizingDyes. The Theory of the Photographic Process, 4th ed.; MacMillan Publishing Co.: New York, 1977;pp 194-234. (6) For a review on the preparation of acetanilidovinyl intermediates: Hamer, F. M. The Cyanine Dyes and Related Compounds. The Chemistry of Heterocyclic Compounds; Interscience Publishers: New York, 1964;Vol. 18,pp 116-147. (7) Piggott, H. A,; Rodd, E. H. Improvements in and Relating to the Manufacture of Intermediates and Dyes from Heterocyclic Nitrogen Compounds. GB Patent 344,409(ICI, La.) 1929; GB 354,898 (ICI, Ltd.), 1930. (8) Stetsenko, A. V.; Yarmolyuk, S. N. Benzoimidacyanines. Ukr. Khim. Zh. (Russ. Ed.) 1981,47 (9),964-966. (9) Rozhinskii, Y. I.; Mostoslavskii, M. A. N-Substituted Naphthostyryl Derivatives. I. Synthesis of N-Alkyl-naphthostyryls. Zh.Org. Khim. 1972,8,2177-2181. (10) Mikhailenko, F.A.;Vasilenko, N. P.; Kachkovskii, A. D.; Rozhinskii, Y.I. Effect of Polar Substituents and the Length of the Polymethine Chain on the Color of Cyanine Dyes of the Benzo[c,d]indole Series. Zh.Org. Khim. 1982,18,435-441. (11) Brooker, L. G. S.; VanLare, E. Cyanine Dyes. US Patent 3,431,111(Eastman Kodak Company) 1969. (12) Kalenda, N. W.Supersensitive Photographic Silver Halide Emulsions. US Patent 3,173,791(1965). (13) Mosby, W. L. Reactions of 8-Valerolactone with ortho- and peri-Naphthylenediamines.J. Org. Chem. 1959,24,414.121.
0 1992 American Chemical Society
*-
1274 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 7
Lawrence et al.
Table I. Physical and Biochemical Resulta of Unsymmetrical Naphth[2,3-d]imidazoliumDerivatives
Y-
no.
R
Y-
6d
Et
I
31
mp, OC 285-86
6e
Et
I
39
265-66
320
* 30
7a
Et
c1
95.6
236-38
390
* 6ZC
7b
Et
c1
88.2
160-63
360
* 60
7c
CHzCHzOAc
c1
51.6
200-02
410
23
7d
CHZCHZOH
c1
31.6
226-28
450
* 52
9
Et
C1
71.4
265-68
2000 i 120
Z
% yield'
formula
IC&): nM 310 i 9
*
substance P 0.12 0.06 "Yield of last step. bConcentration necessary for 50% inhibition (IC,) are means SEM for three determinanta unless otherwise noted. See Experimental Section for procedure. CSameas in footnote b with the exception that four determinanta were used in the calculation of ICW. 0.3 0
s,P E"
Figure 1. Generic structure for imidazo[4,5b]quinoxaJjniumand naphth[2,3-d]imidazolium quaternary salts.
-
-
0.2
L
I. 2 = N,Imidazo[4,5-blquinoxalinederivatives 11.2 = C, Naphth[2,3-dlimidazolederivatives R, R = alkyl,X' = chloride and iodide
Kd = 0.15 nM; Bmax = 37 fmol/mg Compound 78 (130 nM) Kd = 0.23 nM; Bmax 41 fmol/mg Compound78 (260 nM) Kd = 0.30 nM; Bmax = 38 fmol/mg Compound7a (390 nM) Kd 0.40 nM; Bmax = 41 fmol/mg
Qa E
IL
3
0.1
C
2 0.0
an alkyl halide or t0sy1ate.l~ Biological Results and Discussion Target compounds were evaluated in a NK-1receptor The reversibility binding assay using rat forebrain
Figure 2. lBI-BH-substance P binding to rat forebrain with 78. Scatchard analysis.
(14) Lincoln,L. L.; Brooker, L. G. S. Photographic SensitizingDyes of the Merocyanine and Styryl Types in Silver Halide Photographic Emulsions. US Patent 3,397,981 (Eastman Kodak Company), 1968. (15) Park, C. H.; Massari, V. J.; Quirion, R.; Tizabi, Y.; Shultz, C. W.;O'Donohue, T. L. Characteristics of 3H-Substance P Binding Sites in Rat Brain Membranes. Peptides 1984, 5, 833-836.
of the compound-receptor interaction was established by Scatchard analyses. The results of this analysis for compound 7a indicated a concentration-dependent increase in the apparent Kd for SP with no change in,B (Figure 2). This suggests that there is a fully reversible interaction of 7a with the NK-1receptor. To further investigate the nature of the interaction of 7a with the NK-1receptor, the dissociation rate of SP was investigated in the presence
0
10
20 Bound (fmol/mg)
30
40
Substance P Antagonist Naphthimidazolium Derivatives
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 7 1275
Scheme 1"
2
-
loo 120%
DBU,W p l , reflux
E.:::::., A@, reflux 2) ion oxchmge
(a) Ion exchange. Regression equation: - 0.9 pA2 7.4
-
1.8 -
X
-
6.8
0
0.2I 5.9
1
I
I
I
I
6.1
6.3
6.5
6.7
6.9
1
-Log PI (where [E] is the antagonist molar concentration) Figure 4. Schild plot of compound 7a. -7
-6 Log Substance P Concentration -10
-9
-8
Figure 3. Effect of compound 7a on substance P induced contractility of isolated guinea pig ileum.
and absence of test compound 7a (data not shown). In the presence of 2.5 and 10 pM compound 7a, the rate of dissociation was increased by 1.3- and l.bfold, respectively, when compared to the resulta in the absence of test compound. This suggests either the interaction of 7a with the receptor is not strictly competitive or that some nons@ic interaction is occurring at these relatively high concentrations of test compound. The structure-activity relationships observed in this class of compounds were based on potency in the receptor binding assay (Tables I and 11). The nonbridged na-
phthimidazoliums (6d,e and 7a-d, Table I) showed comparable binding potencies. Thus,introduction of hydrogen bonding substituents within the naphth[2,3-d]imidazole nucleus or changing the heterocycle on the "right-hand portion" of the molecule had no effect on binding potency. However, incorporation of a bulky benz[cd]indole moiety as in 9 resulted in an approximately 4-fold decrease in binding potency. Within the 1,3-diethyl imidazoquinoxaline aeries,a 2-fold decrease in binding potency had occurred when the benz[cd]indole nucleus was incorporated into the molecule? Within the bridged naphth[2,3-d]imidazoleseries (Table 111, the benzothiazole heterocycle (134 was the most potent in binding with an ICm = 180 nM. Analogues 13a, 13b, and 13d showed only marginal activity. To determine agonist/antagonist activity of this class of compounds, 7a was selected for further evaluation in
Lawrence et 01.
1276 Journal of Medicinal Chemistry, 1992, Vol. 35, No.7
Scheme I1 R
r 5
Y-
13 12
Table 11. Physical and Biochemical Results of Unsymmetrical Bridged Naphth[2,3-d]imidazolocarbocyanineDyes
Z
no. 13a
Me
Me
R
n
% yield"
mp, 'C
H
1
44
H
1
Me
formula
ICm) nM
283-284
CZeHdN3
790 t 15oC
63
219-221
CnHaIN30
4200 f 850
1
54
278-279
CBHBIN3S
180 f 15
H
2
36
281-282
CBHBINSS
1300 f 180
Me
1
10
292-293
CsrHuIN3
310 t 56
I
Me
13b I
Et
13c I
El
13d
I
Et
13e
I
Me
substance P Yield of last step.
0.12 f 0.06 b*
See Table I for explanation of footnote.
a guinea pig ileum ( g ~ icontractility ) ~ s s ~ Y . ' ~No agonist activity was observed with this compound. Tissue pretreatment With 7a caused a dose-dependent (10-8 M to 10-a M) parallel right shift in the SP dose-response curve (Figure 3). Higher concentrations produced progressive (16) VanRoaaum, J. M. Cumulative Dose-Response Curves 11. Techniques for the Making of Dose-Response Curves in Isolated Organs and the Evaluation of Drug Parameters. Znt. Pharmacodyn. 1963,143,29+330.
Table III. GPI Contractility and in vivo Data PA2 rat salivation: compd Icmb (95% CL) slope EDm (mg/kg, iv) ,a 390 f 62c 7.45 (6.66-8.05) -0.92 0.48 140 510 f lood 7.23 (6.84-7.62) -1.06 1.73 spantide 500 t 18 6.37 (6.04-6.70) -0.95 1.25 "Figure 1: I; imidazoquinoxallne analogue of 7a. bSee Table I for explanation of footnote. 'See Table I for explanation of footnote. dSame as footnote c with the exception that five determinants were used in the calculation of ICw
Substance P Antagonist Naphthimidazolium Derivatives
decreases in E,. The pA2 value was calculated from Schild analyses (Figure 4) of the contractility data, and the results are summarized in Table III. This pA2of 7.42 for compound 7a is more potent than the calculated pA, based on its binding potency. Thus, the ICMof 7a (390 f 31 nM), which corresponds to a Ki of 195 (50% of ICM), correlates to a calculated pA2 value of 6.71. With 95% confidence limits, these PA, values differ in potency only by approximately 5-fold. This discrepancy may be due to the basic differences of these two assays like whole tissue/membrane fractions, neuronal/non-neuronal tissue, and physiological/nonphysiologicalbuffers. Compound 7a, with a pA2of 7.42, was as potent an antagonist as the corresponding imidazoquinoxaline 14 (PA, = 7.23) and was more potent than the reference peptide antagonist spantide'? (pA2 = 6.37). The in vivo SP antagonist activity of 7a was determined in rats using the SP-induced salivationl8and paw edema assays.1g The EDM'sof this compound (7a),the corresponding imidazoquinoxalineanalogue (141, and spantide are given in Table 111. From the data, compound 7a was found to be more potent than both spantide and 14 in inhibiting SP-induced salivation. In addition, 7a was found to be active in inhibiting SP-induced paw edema following PO administration. The minimum effective dose (MED) for 7a in the edema assay was 31.6 mg/kg. This was comparable to the MED obtained for the imidazoquinoxaline analogue 14 (31.6 mg/kg). Further investigation of in vivo SP antagonist activity of 7a revealed that during intravenous (iv) administration at d m of 3 mg/kg, the animals experienced losses of muscle tone, dyspnea, and cyanosis as toxicological effects. Conclusions The biochemical and pharmacological data obtained for the naphth-123-imidazoliumsindicate that they are NK-1 receptor antagonists. This was confirmed by their inhibitory activities in SP-induced salivation and paw edema assays. Even though their binding potency and tissue contractility were comparable to the corresponding imidazoquin~xalinee~ I (Figure l),these compounds were approximately 4-fold more potent in the rat salivation assay (Table 111). Due to the apparent toxic effects of 7a in physiologicalmodels at doses only slightly higher than the effective pharmacological doses, the therapeutic relevance of this class of SP antagonists could not be assessed. As a result, no further work is currently planned within this series of compounds. Experimental Section Melting points were recorded using a Thomas-Hoover or Mel-Temp melting point apparatus and are uncorrected. Proton NMR spectra were obtained in either deuterated dimethyl sulfoxide or deuterated chloroform using a General Electric QE-300 spectrometer. Chemical shifts were reported in ppm downfield from internal standard (tetramethylsilane). Elemental analyses were obtained on all intermediates and are within *0.4% unless otherwise noted. Final compounds were difficult to analyze by combustion analysismand were characterized from spectrwopic (17) Folkers, K.; Hakanson, R.;Horig, J.; Jie-Cheng, X.; Leander,
S. Biological Evaluation of Substance P Antagonists. Br. J. Pharmacol. 1984,83,449-456. (18) Wagner, L. E.; Tomczuk, B. E.; Yanni, J. M. Measurement of Tachykmin-Induced Salivation in Conscious Rata. J. Pharmcol. Methods 1991,26, 67-72. (19) Devor, M.; Papir-Kricheli, D.; Nachmias, E.; Rosenthal, F.; Gilon, C.; Chorev, M.; Selinger, Z.Substance P Induced Cutaneous Plasma Extravasation in Rata is Mediated by NK-1 Tachykinin Receptors. Neurosci. Lett. 1989, 103, 203-208. (20) Link, S. unpublished results, Eastman Kodak Company.
Journal of Medicinal Chemistry, 1992, Vol. 35,No. 7 1277 data only (proton NMR, FDMS, and UV-vis spectroscopy),and these were found to be homogeneous by HPLC. Mass spectra were obtained in either electron impact (EI) or field desorption (FD) modes. Ultraviolet-visible spectra for final dyes were obtained in acetonitrile (HPLC grade, Baker) using a Perkin-Elmer h b d a 9 W/vis/nirspectrophotometer. HPLC retention times were obtained using either a Hewlett-Packard 1090 liquid chromatograph or a Waters 990 3D-Chromatograph. The HewlettPackard 1090 liquid chromatograph was equipped with a 25-cm Kodak Supelco column (supelcosil,LC-8), a programmable solvent gradient system and a Hewlett-Packard diode array detector. A tetrabutylammonium phosphate ion pair buffer system served as the mobile phase. Retention times are given for an eluent flow rate of 1.5 mL/min. The Waters 990 3D-Chromatograph was equipped with a 15-cm Hamilton PRP-1column, a programmable solvent gradient system, and a Hewlett-Packard diode array detector. An ammonium acetate buffer system (pH = 4.65) was used as the mobile phase. Retention times are given for an eluent flow rate of 2.0 mL/min. All reactions were carried out under nitrogen atmosphere. Anhydrous magnesium sulfate was used as a drying reagent, and solutions were concentrated using a rotary evaporator. 2-Methyl-lEI-naphth[2,3-d]imidazole (2). To a stirred solution of 6.32 g (40.0 mmol) of 2,3-diaminonaphthalene(1) in 80 mL of NJV-dimethylformamide, 8.43 g (52.0 mmol) of triethyl orthoacetate was added dropwise over a 3-min period. The reaction mixture was then treated with a catalytic amount of concentrated sulfuric acid (0.16 mL) and heated at 120 "C for 2 h. Heating was continued at 140 "C for 2 h. The resulting brown solution was cooled to room temperature, poured into 200 mL of ice/water, and neutralized to a pH between 7 and 8 using saturated sodium bicarbonate solution. The precipitated solid was collected by filtration, washed with water, and dried under vacuum yielding 6.92 g of a tan solid. This solid was slurried in 250 mL of boiling dichloromethane and filtered hot, followed by stirring in b o i i acetonitrile. The rose-colored solid was collected by filtration and dried to yield 5.0 g (68.7%) of 2: mp 275 "C dec (lit.21mp 286 "C). Anal. (C12HloN2-0.1H20) C, H, N. l-Ethyl-2-methyl-1EI-naphth[2,3-d]imidazole (3). To a stirred suspension of 5.61 g (100.0 mmol) of potassium hydroxide in 100 mL of acetone was added 3.64 g (20.0 m o l ) of 2 in portions, and s t i r r i i was continued at room temperature until the reaction mixture became homogeneous (30 min). The reaction mixture was treated dropwise with 1.7 mL (22.0 mmol) of iodoethane and stirred continually at room temperature for 3 h. The insolubles were removed by fitration, washed thoroughly with acetone, and the combined fdtrates were concentrated. The oily brown residue was treated with 200 mL of water and extracted with ethyl acetate (3 X 75 mL). The combined organic layers were washed with saturated sodium chloride, dried, filtered, and concentrated. Crystallization from anhydrous ether yielded 2.26 g (53.8%) of a light brown solid (3): mp 91-93 "C (lit.21mp 52 "C). Anal. (C1~H1~N2.0.1H20) C, H, N. 1,3-Diethyl-2-rnethyl-lH-naphth[2,3-d]imidazolium Iodide (4a). A solution of 2.10 g (10.0 mmol) of 3 and 15 mL of iodoethane was heated to 100 "C in an oil bath for 4 h. The slurry was cooled to room temperature and treated with 30 mL of anhydrous ether. The resulting solid was collected by filtration and dried overnight, yielding 3.45 g (94.3%) of a pale yellow solid (4a): mp 295 "C dec; 'H NMR (DMSO) 6 1.46 (t, 3 H), 3.02 (s,3 H), 4.58 (9, 2 H), 7.61 (m, 2 H), 8.13 (m, 2 H), 8.60 (8, 2 H). Anal. (CieHisW C, H,N. l-Ethyl-3-(2-hydroxyethyl)-2-met hyl-la-naphth[ 2,3-d]imidazolium Iodide (4b). This quaternary salt was prepared as described for 4a with the following modifications: freshly distilled 2-iodoethanol was used, reaction temperature was 120 "C, final reaction mixture was slurried in acetone yielding a white solid (4b) (26.9%): mp 288-290 "C; 'H NMR (DMSO) 6 1.46 (t, 3 H), 3.00 (8, 3 H), 3.88 (m, 2 H), 4.61 (m, 4 H), 5.11 (t br, 1 H), 7.63 (m, 2 H), 8.15 (m, 2 H), 8.57 ( 8 , 1 H), 8.62 (s, 1 H). Anal. (CieHisIN20) C, H, N.
R.;Schilling, K. Tricyclic Compounds in Which Naphthalene Is Joined by "ortho Fusion" with a Heterocyclic Compound. Ann. Chim. 1935,516, 24Ei-285.
(21) Fries, K.; Walter,
1278 Journal of Medicinal Chemistry, 1992, Vol. 35, No.7
Lawrence et al.
1,3-Diethyl-2-[3-(1,3-dihydro-l,3,3-trimethyl-2H-ind01-2- indolium iodide?was added in 1 portion followed by the dropwise addition of 0.10 g (0.66 mmol) of DBU. The resulting red-orange y1idene)-1-propenyl]- lH-naphth[2,3-d]imidazoliumIodide solution was heated at reflux for an additional 1.5 h and then (6a). A slurry of 0.55 g (1.5mmol) of 4a and 0.67 g (1.5mmol) cooled to 0 OC. The precipitated solid was collected by filtration, of 2-(2-acetanilidoviny1)-1,3,3-trimethyl-3H-indolium iodide? in washed with anhydrous ether, and dried under vacuum, yielding 10 mL of acetonitrile was heated to reflux and treated dropwise 0.12 g (66.7%) of a fluffy, orange solid (6c): mp 248-250 "C; 'H with 0.27 g (1.8 mmol) of DBU, yielding a bright red-orange, NMR (DMSO) 6 1.60 (m, 9 H), 1.83 (a, 3 H), 3.38 (a, 1 H), 4.53 homogeneous solution. The reaction mixture was heated at reflux (br m, 4 H), 4.77 (br t, 2 H), 6.03 (d, 1 H), 6.52 (d, 1 H), 7.03 (t, for 30 min and then cooled to 0 "C overnight. The precipitated 1H), 7.13 (d, 1 H), 7.28 (t, 1 H), 7.43 (a, 1H), 7.77 (m, 2 H),7.98 solid was collected by filtration, washed thoroughly with ether, (t, 1 H), 8.10 (br m, 2 H), 8.38 (a, 1 H), 8.41 (a, 1 H); MS m / e 480, and dried, yielding 0.70 g (85.4%) of a fluffy, red solid (6a): mp HPLC retention time = 10.75 rnin (Waters system). 219-221 "C; 'H NMR (DMSO) 6 1.54 (t, 6 H, 7.15 Hz), 1.61 (a, 3- (2-Acetoxyet hy1)- 1-ethyl-243-( 1,3-dihydro- 1,3,3-tri6 H), 3.38 (a, 3 H), 4.53 (q,4H, 7.25 Hz), 6.07 (d, 1H, 12.48Hz), methyl-2H-indol-2-ylidene)l-propenyl]-lH-naphth[2,3-d]6.49 (d, 1 H, 14.6 Hz), 7.04 (t, 1 H, 7.29 Hz), 7.13 (d, 1 H, 7.68 imidazolium Chloride (7c) a n d l-Ethyl-3-(2-hydroxyHz), 7.29(t, 1 H, 7.69 Hz), 7.43 (d, 1 H, 7.47 Hz), 7.56 (m, 2 HI, ethyl)-2-[3-( 1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylid7.95 (t, 1 H, 13.5 Hz), 8.09 (m,2 H), 8.39 (a, 2 H); MS m / e 422; ene)-l-propenyl]-lH-naphth[2,3-d]imidazoliumChloride UV X, (CHsCN) = 502.2 nM, t (CHsCN) = 7.60 X 10'. 1,3-Diethyl-2-[3-(1,3-dihydro-3,3-dimethyl-l-ethyl-2H-in- (7d). Ion exchange of 6c was achieved using the procedure dol-2-ylidene)-l-propenyl]-l~-naphth[2,3-d]imidazolium outlined for 7a. Proton NMR of the crude product showed it to be a 21 mixture of acylated and nonacylated dyes, respectively. Iodide (6b). To a stirred solution of 0.21 g (0.5 mmol) of 2-(2This mixture (0.55g, 1.2 mmol) was slurried in 8 mL of acetoacetanilidovinyl)-3,3-dimethyl-l-ethyl-3H-indolium iodide' in 5 nitrile, heated to reflux, and treated dropwise with 0.11 mL (1.2 mL of acetonitrile 0.14 mL (1.5mmol) of acetic anhydride was mmol) of acetic anhydride. The resulting mixture was heated added. The resulting mixture was heated to reflux for 3 h. An at reflux for an additional 1 h and then cooled to 0 "C. The additional 0.09 mL (1.0mmol) of acetic anhydride was added, precipitated solid was collected by filtration, washed with ether, and heating was continued for 1 h. The reaction was cooled to and dried, yielding 0.18g (31.6%) of a fme orange solid (7d): mp room temperature overnight, yielding the crude acetanilidovinyl 226-228 "C; 'H NMR (DMSO) 13 1.53 (t, 3 H), 1.61 (8, 6 HI, 3.38 intermediate? To this solution 0.18g (0.5 mmol) of 4a was added (a, 3 H), 4.06 (br m, 2 H), 4.55 (br m, 4 H), 5.48 (br m, 1 H), 6.02 in 1 portion. The mixture was heated to reflux, treated dropwise (d, 1 H), 6.48 (d, 1 H), 7.03 (t, 1 H), 7.12 (d, 1 H), 7.27 (t, 1 H), with 0.09 g (0.6 mmol) of DBU, and heated at reflux for an 7.43 (d, 1 H), 7.57 (m, 2 H), 8.10 (br m, 2 H), 8.38 (m, 3 H); HPLC additional 1.5 h. The reaction mixture was cooled in a refrigerator retention time = 13.56 min (HewletbPackard system). The filtrate overnight. The resulting solid was collected by filtration and was concentrated, and the residue was redissolved in 10 mL of washed with cold acetonitrile, followed by ether to yield 0.22 g acetonitrile and treated with 3 drops of DBU to decompose any (78.6%) of a fluffy, red-orange solid (6b): mp 237-239 "C; 'H excess acetic anhydride. The solution was then treated with NMR (DMSO) 6 1.24(t, 3 H, 6.96 Hz), 1.55 (t,6 H, 7.11 Hz),1.61 anhydrous ether to the cloud point, chilled, and filtered, yielding (a, 6 H), 3.93 (br q, 2 H, 6.99 Hz), 4.52 (br q, 4 H, 7.04 Hz), 6.15 0.32 g (51.6%) of a fine orange solid (70): mp 200-202 "C; 'H (d, 1 H, 12.32 Hz), 6.52 (d, 1 H, 14.58 Hz), 7.05 (t,1 H, 7.43 Hz), NMR (DMSO) 6 1.60 (m, 9 H), 1.83 (a, 3 H), 3.40 (a, 3 H), 4.53 7.13 (d, 1 H, 8.01 Hz), 7.29 (t, 1 H, 7.67 Hz), 7.44 (d, 1 H, 7.57 (br m, 4 H), 4.78 (br t, 2 H), 6.07 (d, 1 H), 6.53 (d, 1 H), 7.05 (t, Hz), 7.55 (m, 2 H), 7.95 (t, 1 H, 13.54 Hz), 8.09 (m, 2 H), 8.38 (a, 1 H), 7.15 (d, 1 H), 7.30 (t, 1H), 7.45 (d, 1 H), 7.57 (m, 2 HI, 7.98 2 H); MS m e 436;UV ,A, (CH3CN) = 501.2 nm, c (CH3CN) = 7.78 X 10. (t, 1 H), 8.10 (br m, 2 H), 8.40 (a, 1 H), 8.42 (a, 1 H);HPLC 1,3-Diethyl-2-[3-(1,3-dihydro-l,3,3-trimethy1-2H-indol-2- retention time = 16.83 min (Hewlett-Packard system). 1,3-Diethyl-2-[3-( l-methylbenz[cd]indol-2(l a ) - y l i d ylidene)-l-propenyl]-lH-naphth[2,3-d]imidaazoliumChloride ene)-1-propeny11-l a - n a p h th[ 23d]imidazolium Chloride (9). (7a). A solution of 1.1 g (2.0 mmol) of 6a in 75 mL of methanol A slurry of 0.31 g (1.5 mmol) of [1-methylbenz[cd]indol-2was treated with 11.0 g of amberlite IRA-400 chloride ion-exchange (lH)-ylidene]acetaldehyde (8)*11 and 1,3-diethy1-2-methyl-lHresin, and the mixture was allowed to stir at room temperature naphth[2,3-~~daz#liumiodide (4a)in 10 mL of acetic anhydride for 1 h. The resin was removed by filtration and washed with was stirred at 130 "C in an oil bath for 1.5 h. The resulting mixture methanol. The combined filtrates were treated with 11.0g of freah was cooled to room temperature, and the precipitated solid was resin and stirred for 1 h. This process of stirring and filtering collected by filtration, dried, and recrystallized from 50 mL was repeated two times. Finally, the filtrate was concentrated, ethanol, yielding 0.53 g (63%) of a dark red solid. This iodide yielding crude 7a which was purified by suspending the solid in salt was converted to the corresponding chloride salt via the hot toluene and filtering to yield 0.88 g (95.6%) of a fme red solid procedure outlined for 7a. Crystallization induced from etha(7a): mp 236238 "C;'H NMR (DMSO) S 1.54 (t, 6 H, 7.13), 1.61 nol/ether yielded 0.30g (71.4%) of a fme, dark blue solid (9): mp (a, 6 H), 3.39 (a, 3 H), 4.53 (br q,4 H, 7.05 Hz), 6.08(d, 1 H, 12.47 265-268 OC dec; 'H NMR (DMSO) 6 1.60 (t, 6 H), 3.58 (a, 3 H), Hz), 6.51 (d, 1 H, 14.54 Hz), 7.04 (t, 1 H, 7.36 Hz), 7.13 (d, 1 H, 4.63 (br m, 4 H), 6.58 (d, 1 H), 6.93 (d, 1 H), 7.10 (d, 1 H), 7.46 7.85 Hz), 7.28 (t, 1 H, 7.69 Hz), 7.43 (d, 1 H, 7.13 Hz), 7.56 (m, (m, 2 H), 7.60 (m, 2 H), 7.77 (t, 2 H), 7.97 (d, 1 H), 8.13 (m, 3 H), 2 H), 7.94 (t, 1 H, 13.53 Hz), 8.08 (m, 2 H), 8.39 (a, 2 H); MS m / e 8.33 (t, 1 H), 8.50 (a, 1 H); MS m / e 430; HPLC retention time (CH,CN) = 498.8 nm, t (CH3CN) = 7.56 X lo4. 422;UV ,A, = 24.38 min (Hewlett-Packard system). HPLC retention time = 24.57 min (Hewlett-Packard system). Substance P Receptor Binding Method. 1261-Bolton l,3-Diet hyl-24 3-( 1,3-dihydro-3,3-dimethyl-l-ethyl-2H-inHunter-Substance P ('%I-BH-SP2200 Ci/mmol) was purchased dol-2-y1idene)-l-propenyl]-lH-naphth[2,3-d]imidazolium from New England Nuclear. Receptor binding was performed Chloride (7b). This compound was prepared by the method according to Park et al.15 outlined for 7a and was recrystallid from acetonitde/anhydroUs The forebrain (whole brain minus cerebellum) of a male ether, yielding 0.75 g (88.2%) of a red solid (7b): mp 160-163 Spragw-Dawley rat was homogenized in 20 volumes of ice-cold "C; 'H NMR (DMSO) 6 1.24 (t, 3 H, 7 Hz), 1.55 (t, 6 H, 7.12 Hz), 50 mM tris(hydroxymethy1)aminomethane hydrochloride 1.61 (a, 6 H), 3.93 (br q, 2 H, 7.12 Hz), 4.52 (br q, 4 H, 6.96 Hz), (TrisaHCl),pH 7.4,at 4 "C, 5 mM KCl, and 120 mM NaCl (wash 6.15 (d, 1 H, 12.4 Hz), 6.52 (d, 1 H, 14.5 Hz), 7.05 (t, 1 H,7.4 Hz), buffer) with a Tekmar Tissumizer Mark II. The homogenate was 7.13(d, 1 H, 7.9 Hz),7.29 (t, 1 H, 7.71 Hz), 7.44 (d, 1 H, 7.2 Hz), centrifuged at 48,OOOgfor 10 min, and then the resulting pellet 7.56 (m, 2 H), 7.95 (t, 1 H, 12.8 Hz), 8.08 (m, 2 H), 8.38 (e, 2 H); was resuspended in 50 mM Tris-HC1, pH 7.4,at 4 "C, 10 mM MS m / e 436;UV A, (CHsCN) = 502.2 nm, c (CH3CN)= 7.60 ethylenediaminetetraaceticacid (EDTA), and 300 mM KC1, and X lo4;HPLC retention time = 11.14 min (Waters system). incubated for 30 min at 4 "C. The homogenate was then cen3-(2-Acetoxyethyl)-l-ethyl-2-[3-( 1,3-dihydro-1,3,3-trimethyl-2H-indol-2-y~dene)-l-propenyl]-lH-naphth[2,3-d]-trifuged as above and centrifuged twice more in 50 mM TriiHC1 (pH 7.4)at 25 "C. The final pellet was resuspended in 60 volumes imidazolium Iodide (6c). To a stirred sluny of 0.11 g (0.3mmol) of Tris buffer. of 4b in 3 mL of acetonitrile, 0.06 g (0.6mmol) of acetic anhydride The binding assay mixture, 0.25 mL, contained 75-125 pg of was added in 1 portion. The resulting mixture was heated at reflux membrane protein, 0.1 nM '%I-BH-SP, test compound in 50 mM until the reaction mixture became homogeneous. At this point 0.13 g (0.3mmol) of 2-(2-acetanilidoviny1)-1,3,3-trimethyl-3H- Tris.HC1, pH 7.4 at 25 "C, 0.02% bovine rum albumin, 2 rcg/mL
I
Substance P Antagonist Naphthimidazolium Derivatives chymostatin, 4 pg/mL leupeptin, 40 pg/mL bacitracin, and 1.2 mM MnC12. Nonspecific biding was defied with 1 pM SP. All assays were run in duplicate or triplicate, and the reaction mixtures were incubated 20 min at 25 “C. The assay mixtures were then diluted with 2 mL of wash buffer and fitered through GF/C glass fiber filters presoaked in 0.01% polyethylenimine. The filters were washed eight times more with 2 mL of wash buffer. The radioactivity trapped on the filters was counted in a Packard Cobra gamma counter. The competition curve data were analyzed by computer nonlinear least squares best fit of the data to the Hill equation which determines the ICmvalues from at least seven M to 1 X lod M). concentrationsof the test compound (1 X Isolated Guinea Pig Ileum Contractility Method. Hartley guinea pigs of either sex (250-500 g) were killed by C02inhalation. The point at which the terminal ileum enters the cecum was located. The ileum was cut, and a section approximately 30 cm in length was removed. The ileum was dissected free of fat and connective tissue, and the terminal 5 cm was discarded. Individual sections, approximately 2.0 cm in length, were then cut beginniig at the terminal end and suspended in a jacketed, 10-mL, siliconized, glass organ bath containing Tyrode’s solution of the following composition in m M 136.9 NaC1, 2.7 KCl, 0.98 MgClZ, 1.80 CaCl,, 0.47 NaHZPO4H20,11.9 NaHC03, 5.5 dextrose. The Tyrode’s solution was supplemented with lo4 M indomethacin, pyrilamine, and atropine, and bacitracin (40 mg/L). The tissues were maintained at 37 OC and gassed with 95% 02-5% C02. A resting tension of 1 g was applied to the tissue, and the preparations were allowed to equilibrate for 60 rnin with washes every 15 min. Isometric contractions were recorded using Grass FT-03 force displacement transducers attached to a Grass Model 7E polygraph. A submaximal concentration (10 nM) of the agonist SP was then added to each tissue, and the individual response was recorded. This was repeated every 15 min until a reproducible response was obtained (usually three to four additions). Thirty minutes following stimulation of the tissues, cumulative doseresponse curves for SP were constructed according to the method of Van Rossum.l8 Tissues were then washed and allowed to restabilize for 30-45 min with washing every 10 min. Test compounds were then added to the tissues 3 min before repeating the cumulative dose-response curves. A shift to the right of the SP dose-response without a significant reduction of the maximal contractile response suggested competitive antagonism of SP by the test compound. Rat Salivation Method. Fed male Sprague-Dawley rats (200-300 g) were weighed and placed into groups of six using random number tables generated by the SAS program, Proc Plan. Conscious rats were injected in vivo, via the lateral tail vein, with either the vehicle (0.9% saline w/v) or sialogogue. The volume of injection for the vehicle and compounds was 1.0 mL/kg. Compounds were injected on a pg/kg basis. The group of rats injected with saline (0.9% w/v) served as the baseline control. The rat’s oral cavity was swabbed immediately after in vivo injection by placing and holding a preweighed absorbant foam cube (5/16 in., Texwipe Company, Upper Saddle River, NJ)
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 7 1279 sublingually for 10 sec using a Triceps foam pencil (Texwipe Company, Upper Saddle River, NJ). Foam cubes were reweighed immediately after use. The difference between the initial weight of the cube and the weight of the cube after use represents saliva secreted. Data were analyzed with Dunnett’s t-testz2 that compares several treated groups with a control group. Regression analysis was used to determine doseresponse and relative potency.29 Substance P Induced Paw Edema Method. Fasted male Sprague-Dawley rats were randomized into groups of six. The volume of the hind paw of each rat was determined plethysmographically by means of a mercury filled vessel connected to a pressure transducer (Grass p23A) that in turn was connected to strip chart recorder (linear, Cole Parmer Model No. 55). The instrument was calibrated so that 1 mL of fluid produced a pen deflection of 25 mm. The test compounds were dissolved or suspended in aqueous 0.5% Tween 80. Sonification was used to facilitate solubilizationor reduce particle size. Rats were dosed orally with the tween 80 vehicle or test compounds 1 h prior to the subcutaneous injection of 0.1 mL of a solution containing 100 pg/mL of SP (Cambridge Research Biochemicals, Cambridge, MA) in 0.9% saliie. Fifteen minutes after SP injection the hind paw volume was measured as described, and edema was determined by the difference in the two volume measurements. Results were expressed as edema (mL). The data were analyzed with Dunnett’s t-test?2 Regression analysis was used to determine dose-response.23 The protocol for this experiment was reviewed by the animal Care review committee at Eastman Kodak Co. and all animal care and use associated with this procedure meets NIH guidelines.
Acknowledgment. We would like to thank Lewis Lincoln and Earl VanLare of Eastman Kodak Co. for the preparation of all the bridged naphth[ 2,&d]imidazolium derivatives and intermediates. We also acknowledge the help of Barbara Fragale, Colleen Duggan, and Cheryl Meravi with the biochemical evaluation. We thank Larry Wagner for the pharmacological evaluation and the members of the Dye Research Laboratories at Eastman Kodak Co. for valuable discussions. Registry No. 1, 771-97-1; 2, 1792-36-5; 3, 80079-34-1; 4a, 139167-41-2; 4b, 139167-42-3;5,40497-16-3;6a, 139167-43-4; 6b, 139167-44-5;6c, 139167-45-6;6d, 80079-28-3;6e, 139167-46-7;7a, 139167-47-8; 7b, 139167-48-9;7c, 139167-49-0;7d, 139167-50-3; 8, 139167-51-4;9, 139167-52-5; 13a, 139167-53-6; 13b, 3055-33-2; 13c, 139167-54-7;13d, 18426-36-3;13e,139167-55-8; substance P, 33507-63-0; 2-(2-acetanilidovinyl)-3,3-dimethyl-l-ethyl-3Hindolium iodide, 139167-56-9. (22) Dunnett, C. W. A Multiple Comparisons Procedure for Comparing Treatments with a Control. J. Am. Stat. Assoc. 1955, 50,1696-1121. (23) Bliss, C. In Vitamin Methods; Gyorgy, E., Ed.;Academic Press: New York, 1951; pp 445-610.
